Field of the Invention
The present disclosure is generally in the technical field of nucleic acid molecule detection that can be implemented, for example on a paper strip or microfluidics device, without nucleic acid extraction or amplification thereof, e.g. via a polymerase chain reaction (PCR). In some embodiments, the detection system can be used substantially near the point of care (POC) so that patients can be treated rapidly and at low cost. More particularly, the present disclosure relates to methods, compositions, systems, and kits for detecting one or more target sequences and, in some embodiments, can be applied in the POC setting. In one aspect, a novel method for detecting and performing multiplex nucleic acid detection without nucleic acid extraction, or amplification thereof are provided. In some embodiments, a sensitive nucleic acid POC diagnostic paper dipstick or microfluidic device is provided that is capable of detecting and identifying all of cancers or all of infectious diseases such as the dengue virus, influenza virus, chikungunya virus, and malaria etc. In some other embodiments, the method is configured such that the target nucleic acid sequence for the method is associated with one or more selected from the group consisting of dengue virus, influenza virus, chikungunya virus, the human immunodeficiency virus (HIV), the Hepatitis C virus (HCV), Human papillomavirus (HPV), Middle East Respiratory Syndrome (MERS) virus, arboviruses, methicillin-resistant staphylococcus aureus (MRSA); a bacterium, a fungus, and a parasite. In some other embodiments, the target nucleic acid sequence is associated with nucleic acid biomarkers selected from the group consisting of RNA, DNA, and microRNA in non-communicable and/or chronic diseases.
Description of the Related Art
A number of non-enzymatic or template mediated chemical ligation methods have been developed that can be used to detect sequence variations. These include chemical ligation methods that utilize coupling reagents, such as N-cyanoimidazole, cyanogen bromide, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. Kool (U.S. Pat. No. 7,033,753), which is incorporated herein by reference in its entirety, describes the use of chemical ligation and fluorescence resonance energy transfer (FRET) to detect genetic polymorphisms. Other chemical ligation methods react a 5′-tosylate or 5′-iodo group with a 3′-phosphorothioate group, resulting in a DNA structure with a sulfur replacing one of the bridging phosphodiester oxygen atoms.
Some of the advantages of using non-enzymatic approaches for nucleic acid target detection include lower sensitivity to non-natural DNA analog structures, ability to use RNA target sequences, lower cost and greater robustness under varied conditions. Letsinger et al. (U.S. Pat. No. 5,780,613, herein incorporated by reference in its entirety) have previously described an irreversible, nonenzymatic, covalent autoligation of adjacent, template-bound oligonucleotides wherein one oligonucleotide has a 5′ displaceable group and the other oligonucleotide has a 3′ thiophosphoryl group.
PCT applications WO 95/15971, PCT/US96/09769, PCT/US97/09739, PCT US99/01705, WO96/40712 and WO98/20162, all of which are expressly incorporated herein by reference in their entirety, describe compositions comprising nucleic acids containing electron transfer moieties, including electrodes, which allow for novel detection methods of nucleic acid hybridization. One technology that has gained increased prominence involves the use of DNA arrays, especially for applications involving simultaneous measurement of numerous nucleic acid targets.